The present invention is directed to a fibrin sealant containing a fibrinolytic inhibitor, a method of isolating such a fibrinolytic inhibitor, and a device that isolates such a fibrinolytic inhibitor through an automated process utilizing centrifugal force.
xe2x80x9cFibrinxe2x80x9d sealants are widely used to reduce bleeding in surgery and to seal blood vessels and tissues that have been dissected either in surgery or through wounding. The term xe2x80x9cfibrinxe2x80x9d can be viewed as a misnomer in this context since historically xe2x80x9cfibrinxe2x80x9d sealants have been delivered as a material containing the precursor of fibrin, namely fibrinogen. In such sealants, fibrinogen material has been co-delivered at the site to be sealed with a proteinase enzyme that converts the fibrinogen to fibrin. Once a sufficient amount of fibrin is formed from the fibrinogen, the fibrin spontaneously polymerizes into a fibrin polymer whichxe2x80x94when sufficient polymer is assembledxe2x80x94forms a fibrin clot. Generally, the conversion enzyme has been bovine thrombin. Recently, however, an effective sealant has been described that delivers fibrin, in a xe2x80x9cfibrin monomerxe2x80x9d form that is stabilized against polymerization, to the site that is to be sealed. At the site, the stabilization conditions are reversed, and an effective clot forms. See, Edwardson et al., European Patent Application No. EP 592,242.
One of the particular advantages of this fibrin monomer sealant of EP 592,242 is that the sealant can be rapidly prepared from a small amount of a patient""s blood only minutes before surgery, and this can be done using standard laboratory equipment. Processes for deriving the fibrinogen material of prior art sealants are much more demanding and more difficult to automate. Specialized tools for preparing fibrin monomer have also recently been described, and these tools allow an autologous sealant to be prepared from a patient in a rapid, highly reproducible, highly reliable, and highly safe manner. See, Holm, xe2x80x9cCentrifuge Reagent Delivery Systemxe2x80x9d, WO 96/16713, Holm et al., xe2x80x9cMethod and Device for Separating Fibrin I from Blood Plasmaxe2x80x9d, WO 96/16714 and Holm, xe2x80x9cCentrifuge with Annular Filterxe2x80x9d, WO 96/16715. These patent applications describe a molded apparatus that operates in a centrifuge. A first chamber of the apparatus is filled with blood, and a centrifugation process separates the plasma from a pelleted cellular blood fraction. The plasma is transferred to a second chamber into which a conversion enzyme, which is covalently bound to biotin, is inserted. The enzyme operates to convert the fibrinogen in the plasma to fibrin, which fibrin molecules bond to one another to form polymers that precipitate to form a solid. The fibrin precipitate is pelleted by centrifugation, and the remaining plasma is transferred back to the first chamber. The pelleted fibrin precipitate is dissolved with a solubilizing liquid, which is most often an aqueous solution buffered at an acidic pH. The viscous fibrin monomer solution is mixed with agarose beads having bound avidin to remove traces of biotinylated conversion enzyme, and then washed into a third chamber (for example, a syringe) through a filter which removes the agarose beads. The retained agarose contains any residual enzyme bound via the high-affinity avidin-biotin interaction. The solubilized fibrin monomer composition can be used as a sealant as described in Edwardson et al., EP 592,242.
These improvements, thus, allow for an autologous sealant to be prepared in a rapid, automated process, and the autologous sealant so prepared is free of extrinsic proteinase enzymes such as bovine thrombin. However, if so prepared, the sealant does not contain quantities of inhibitors against fibrinolysis. These inhibitors can limit the rate, after the sealant has been used to form a fibrin clot, at which the body""s housekeeping enzymes remove clotted fibrin. Aprotinin, a 6200 molecular weight polypeptide isolated from bovine lung, is one such fibrinolytic inhibitor. Bovine aprotinin could, of course, be added to the above-described sealant, but this would partially undermine one of the substantial advantages of the sealant, which advantage is the ability to prepare the sealant autologously so that the sealant and clot formed with the sealant contain only biopolymers that are derived from the patient. In fact, the repeated use in a patient of bovine aprotinin has been associated with adverse consequences such as hypersensitive reactions. Thus, what is needed in the art is a method of easily preparing from the species to be treated, most preferably the patient him or herself, a suitable inhibitor of fibrinolysis. In particular, what is needed is a method that can be coordinated with the automated process described above.
The invention provides a kit for preparing a fibrin sealant, wherein the kit is a first kit (A) comprising: (a) a fibrin monomer preparation; (b) a stabilizing preparation containing a clot-preserving effective amount of a fibrinolysis-inhibiting protein; and (c) a non-enzymatic polymerizing agent preparation effective to convert the fibrin monomer preparation into a fibrin clot; or a second kit (B) comprising: (axe2x80x2) a fibrin monomer preparation comprising a fibrin monomer and a clot-preserving effective amount of a fibrinolysis-inhibiting protein, and (cxe2x80x2) a polymerizing agent preparation effective to convert the fibrin monomer preparation into a fibrin clot. Preferably, the fibrin-clot stabilizing effective amount is: (i) in a stabilizing preparation that is a solution, at least about 70 xcexcg/ml alpha-2-antiplasmin, (more preferably 200 xcexcg/ml, still more preferably 400 xcexcg/ml); or (ii) in a stabilizing preparation that is a solid, at least about 70 mg alpha-2-antiplasmin per g of fibrin, (more preferably 120 mg per g, still more preferably 30 mg per g. Preferably, the fibrin monomer preparation is a liquid and the concentration of fibrin monomer in the preparation is at least about 8 mg/ml (more preferably 15 mg/ml, still more preferably 30 mg/ml). Preferably, the fibrin monomer preparation comprises a fibrin-solubilizing effective amount of acid and the polymerizing agent preparation comprises an amount of base sufficient to bring the amount of acid to less than a fibrin-solubilizing effective amount or the fibrin monomer preparation comprises a fibrin monomer lyophilizate and the polymerizing agent preparation comprises an aqueous buffer.
The invention also provides a method of forming a fibrin sealant from an animal comprising: (a) contacting a first extract from the animal containing a fibrinolysis-inhibiting protein with a clot inhibitor-binding ligand bound to an extraction implement; (b) isolating a first composition comprising a fibrinolysis-inhibiting protein; (c) contacting a second extract from the animal, which contains fibrinogen and which can be the same as the first extract, with a fibrinogen-converting enzyme; and (d) isolating a second composition comprising a clot-forming effective amount of fibrin monomer from the contacted second extract, wherein the amount of isolated fibrinolysis-inhibiting protein is sufficient to stabilize at least a clot-forming effective amount of the second composition. Preferably, the first and second extracts are the same, and the first extract is blood or a blood derivative.
The method can further comprise (1) sealing a tissue to prevent fluid loss or to prevent adhesions to the tissue or (2) coating a material to increase its biocompatibility by: (e) contacting the second composition with a polymerizing agent composition effective to convert the fibrin monomer preparation into a fibrin clot; and (f) further contacting the second composition with a clot preserving effective amount of the first composition.